Poster
MaxCyte Electroporation Enables the Production of Assay-Ready Cells Suitable for Ion Channel Studies
Abstract
Advances in technology for automated electrophysiology have simplified the implementation of high-throughput screening for ion channel research and drug discovery. However, a continued, widespread reliance on stable cell lines remains a bottleneck to progress.
Stable cell line development is a lengthy, laborious process, and creating stable cell lines for ion channel expression can be particularly challenging. Ion channels are complex, multi-subunit proteins, and cell line development may require inducible promoters or multiple rounds of selection with harsh selection agents, potentially altering cell health and growth characteristics.
The MaxCyte® electroporation platform enables the efficient transfection of virtually any cell type for the rapid, scalable transient expression of multi-subunit protein complexes, including toxic or intractable ion channels. Unlike other electroporation methods or chemical or lipid reagents, MaxCyte electroporation delivers high transfection efficiency while maintaining high cell viability and membrane integrity.
MaxCyte transfected cells are suitable for immediate use in cell-based assays, or they can be aliquoted and cryopreserved as stocks of assay-ready cells for later use. MaxCyte’s Flow Electroporation® technology can transfect up to 2x1010 cells in under 30 minutes to produce assay-ready cells in the most appropriate cell type. Here we show scalable, highly efficient transfection in a variety of cell lines and stem cells and present case studies demonstrating the suitability of assay-ready cells as an alternative to stable cell lines for ion channel research, assay development and drug discovery.
MaxCyte electroporation enables use of the most appropriate cell context
High transfection efficiency in virtually any cell type with MaxCyte electroporation
Figure 1: Cells were transfected with 2 μg of pGFP DNA per 1x106 cells using the appropriate pre-loaded MaxCyte electroporation protocol. At 24 hours post transfection, cells were examined for cell viability and transfection efficiency.
*Data courtesy of ixCells.
MaxCyte electroporation is scalable
Consistent assay performance between cells prepared by small- or large-scale electroporation
Figure 2: HEK293F suspension cells were transfected with a GPCR expression plasmid using electroporation at either small scale (4x107 cells) or large scale (1x109 cells). Post electroporation, cells were seeded in 96-well plates at the indicated cells density. GPCR activity was assayed using a cAMP ELISA at 18 hours post transfection. The data demonstrates consistent performance in cells transfected at both small and large scale by MaxCyte electroporation.
MaxCyte transiently transfected cells are suitable for calcium channel studies
A & B) Mean fluorescent signal in K+ stimulated cells treated with a calcium channel antagonist
Figure 3: HEK293 cells were either transfected (A) with the Cav2.2 pore-forming alpha subunit (5-6kb cDNA) and two modulatory subunits, (the modulatory β3 subunit, the modulatory α2δ subunit) and an inward rectifier potassium channel, Kir2.1, or untransfected (B). At 24 hours post electroporation, cells were depolarized with high external potassium, then treated, or untreated, with the calcium channel antagonist, conotoxin and FLIPR® assays were carried out. Together, the results confirm that the signal seen in panel A is due to calcium channel activity, confirming that MaxCyte transiently transfected cells are suitable for calcium channel studies.
Data courtesy of ChanTest, now part of Charles River Laboratories.
High-throughput screening for compounds that restore functional CFTR expression
Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene may disrupt the function of an ion channel involved in hydration maintenance, leading to cystic fibrosis. A stop mutation (G542X) in the CFTR gene is present in around 5% of cystic fibrosis patients. Aminoglycosides, such as G418, promote translational readthrough of non-sense mutations, including the G542X mutation. A high-throughput screening method was developed to identify compounds that promote readthrough of the G542X stop mutation, potentially restoring functional CFTR expression.
A) Preparation of assay-ready cells for rapid assay development
Figure 4: HEK293 cells were transfected with a plasmid encoding a CFTR minigene including the G542X stop mutation. A large stock of matched, validated assay-ready cells was prepared and cryopreserved (A) to enable rapid assay development, optimization, initial validation and miniaturization. Assay-ready cells were used to develop the high-throughput screening (HTS) assay in 1536-well format (B) while a stable cell line was developed.
B) Optimized high-throughput screening assay workflow
C) MaxCyte assay-ready cells outperform lipid transfection
D) Assay miniaturization
E) Comparable performance of assay-ready cells and a stable cell line
Figure 4 (continued): HEK cells transfected with the CFTR G542X minigene using either lipid or MaxCyte electroporation were compared in the initial 384-well assay format. Transfected cells were treated with combinations of PTI-CH (amplifier), VX-809 (corrector) and G418 (positive control) or DMSO (negative control). MaxCyte electroporated cells had a larger assay window than lipid transfected cells (C). MaxCyte electroporation was thus used to prepare assay-ready cells for cost-effective assay development, miniaturization and optimization. Cell titration in 1536-well format using MaxCyte electroporated assay-ready cells demonstrates similar assay response at 2,400 cells per well compared with the initial 384-well format assay (D). Assay-ready cells and a cell line stably expressing the CFTR G542X minigene were tested in the final, optimized assay format. Figure 4E demonstrates consistent performance from both, confirming the suitability of assay-ready cells in this HTS assay.
1Adapted with permission from Smith et al., SLAS Discovery 2021, Vol. 26(2) 205–215. DOI: https://doi.org/10.1177/2472555220962001
Table 1: MaxCyte Flow Electroporation® could enable HTS of the Scripps SDDL
| Screening Round | Cells/Well | Sample # | Doses | Replicates | Total Cell # |
|---|---|---|---|---|---|
| Pilot | 3,600 | 1,280 | 1 | 3 | 1.38x107 |
| Primary | 3,600 | 666,120 | 1 | 3 | 2.40x109 |
| Confirmation | 3,600 | 6,395 | 1 | 3 | 6.91x107 |
| Counter | 3,600 | 6,395 | 1 | 3 | 6.91x107 |
| Titration 1 | 3,600 | 652 | 10 | 3 | 7.04x107 |
| Titration 2 | 3,600 | 652 | 10 | 3 | 7.04x107 |
| Titration 3 | 3,600 | 652 | 10 | 3 | 7.04x107 |
| Total Cell # Needed for HTS Campaign | 2.76x109 |
Table 1: A calculation of the theoretical cell number needed to carry out the full screening campaign, including pilot LOPAC screen, SDDL primary screen, confirmation screening and counter screening, and lead titration. MaxCyte Flow Electroporation with a single CL-2™ processing assembly enables the preparation of up to 2x1010 cells in a single transfection, providing sufficient assay-ready cells for assay development and high-throughput library screening.
MaxCyte electroporation enables high-throughput evaluation of ion channel variants
Kv7 is a family of tetrameric voltage-gated potassium channels encoded by five KCNQ genes expressing KCNQ1-5. The KCNQ2 subunit (Q2) can co-assemble with KCNQ3 (Q3) to form heterotetramers widely expressed in the nervous system. Hundreds of Q2 genetic variants are associated with epilepsy and developmental challenges, but only a fraction of these have been studied. Researchers from the George Lab (Northwestern University) developed and validated a method for the high-throughput functional evaluation of ion channel variants. Here we share their evaluation of 39 previously unstudied epilepsy-associated Q2 variants and their responses to a candidate therapeutic. MaxCyte electroporation was used to produce assay-ready cell banks suitable for automated patch clamping, enabling ion channel variant characterization on an unprecedented scale
A) Workflow for preparation of assay-ready cells and evaluation of KCNQ2 variants
Figure 5: A) CHO-Q3 cells (stably express Q3 subunit) were electroporated with plasmids expressing Q2 variants to generate homozygous (variant Q2 alone) or heterozygous (variant Q2 + WT Q2; 1:1 ratio) assay-ready cell banks, which were cryopreserved until needed. Assay-ready cells were thawed and incubated for 10 hours at 37°C with 5% CO2 then grown overnight at 28°C to increase membrane ion channel expression. Cells were passaged with 5% trypsin then diluted to 200,000 cells/mL in external solution and incubated for 60 minutes at 15°C with shaking. Automated patch clamp recordings were performed.
B) Current density (% WT) v. DV1/2 of activation (mV)
C) Percentage WT control current at -20mV
![A heatmap compares density (percentage of untreated WT under control and retigabine conditions [RTG]) for a list of variants on the x axis. For control, the minimum is coded red and the maximum blue; for retigabine, minimum is yellow and max is green.](https://maxcyte.com/wp-content/uploads/2025/06/Screenshot-2025-06-23-at-11.45.01 AM.png "production-of-assay-ready-cells-suitable-for-ion-channel-studies_fig5-c")
Figure 5 (continued): Differences in peak current density and activation V1/2 for heterozygous variants relative to WT were plotted (B). Most of the heterozygous variants had lower peak current density than WT, indicating loss of function. Figure 5C is a heat map showing the current density of untreated (-) and retigabine-treated (+) KCNQ2 variants as a percentage of untreated WT. Retigabine treatment restored ion channel function to at least WT levels for all the variants tested.
2Content adapted and reproduced from Vanoye et al JCI Insight. 2022 under Creative Commons License Attribution 4.0 International (CC BY 4.0).
Table 2: Summary of ion channels variants
| Channel | Cell Type | Efficiency | EP Protocol | # Constructs | Notes |
|---|---|---|---|---|---|
| KCNQ1 (KV7.1) | CHO (stable KCNE1) | >90% | CHO-PE | 291 | Includes WT co-expression |
| KCNQ2 (KV7.2) | CHO (stable KV7.3) | >90% | CHO-PE | 250 | Includes WT co-expression |
| KCNH1 (KV10.1, eag1) | HEK293T | >90% | 6 | 64 | Includes WT co-expression |
| SCN1A (NaV1.1) | HEK293T | 80-90% | 6 | 61 | Includes 1 splice isoform |
| SCN2A (NaV1.2) | HEK293T | ~70% | 4 | 225 | Includes 2 splice isoforms |
| SCN4A (NaV1.4) | ND7/23-LoNav | 80-95% | ND7 | ||
| SCN5A (NaV1.5) | HEK293T | 85-95% | HEK | ||
| SCN8A (NaV1.6) | ND7/23-LoNav | 75-85% | 4 or 6 | 35 | Includes 2 splice isoforms |
| KCNE1 | 125 | Includes WT co-expression | |||
| KCNB1 (Kv2.1) | 52 | Includes WT co-expression |
Table 2: Summary of ion channels variants (>1,100 constructs) studied by the George Lab (Northwestern University) using MaxCyte electroporated, transiently transfected assay-ready cells. Data reproduced courtesy of Alfred George, Jr., Department of Pharmacology, Northwestern University Feinberg School of Medicine.
Rapid development of assay-ready cells for hNav1.5 studies
A) Workflow for optimizing DNA concentration and cell culture conditions
Figure 6: HEK293 cells were transfected using MaxCyte's scalable electroporation platform with four different concentrations of plasmid, encoding the human Nav1.5 ion channel (courtesy of Thomas Jespersen at Copenhagen University). After transfection, cells were rested for 20-30 minutes then cultured at 37˚C for 24 hours. Half of the cells were then transferred to 28˚C for final culture, while the other half remained at 37°C until harvest. Cells were screened on the Sophion Qpatch II in single hole mode.
Table 3: Optimized DNA concentration and culture temperature
| DNA Concentration (μg/mL) | Temperature (°C) | Transfection Efficiency (%) | Average Current Level (nA)* | TTX block (%)** |
|---|---|---|---|---|
| 100 | 28 | 57 | -6.3 | 65 |
| 150 | 28 | 100 | -5.7 | 71 |
| 200 | 28 | 80 | -3.6 | 82 |
| 250 | 28 | 75 | -3.5 | 86 |
| 100 | 37 | 82 | -2.5 | 77 |
| 150 | 37 | 89 | -2.0 | 79 |
| 200 | 37 | 83 | -3.8 | 79 |
| 250 | 37 | 42 | -3.0 | 82 |
*Measured at 0 mV in simple depolarizing step protocol. **Percentage block compared to saline period of a 20 μM TTX single addition.
Table 3: A DNA concentration of 150 μg/mL was chosen to give optimal results. MaxCyte electroporation efficiently delivered the relatively large Nav1.5 gene to cells which were then suitable for use in automated electrophysiology studies. The current amplitude from cells incubated at 37°C is large enough to enable future studies to be carried out at a physiological temperature, the lower temperature of 28°C consistently produced larger peaks, which might be a useful attribute when studying low expressing channels.
Table 4: Comparison of fresh and cryopreserved assay-ready cells for automated patch clamp screening
| Freshly Electroporated Cells | Cryopreserved Cells | |||
|---|---|---|---|---|
| Temperature (°C) | Average Current Level (nA)* | TTX Block (%)** | Average Current Level (nA)* | TTX block (%)** |
| 28 | -6.5 | 85 | -5.4 | 81 |
| 37 | -5.2 | 85 | -4.8 | 79 |
*Measured at 0 mV in simple depolarizing step protocol. **Percentage block compared to saline period of a 20 μM TTX single addition.
Table 4: HEK293 cells (four samples of 4x107cells) were electroporated with 150 μg/ml of the Nav1.5 expression construct using the MaxCyte scalable electroporation system. After resting, transfected cells were cultured in four T175 flasks at 37°C for 24 hours. Two flasks were then transferred to 28°C and two flasks were maintaining at 37°C. After 48 hours in final culture, the cells were tested on the Qpatch II or aliquoted and frozen down according to Sophion’s recommended cryopreservation strategy (QGUIDE 16153). The frozen cells were subsequently revived and tested on the Qpatch II. The overall success rate on the Qpatch II was 61% for the “fresh” cells and 60% for the “frozen” cells effectively showing no significant difference.
Conclusion
This study demonstrates that MaxCyte electroporation produces efficiently transfected cells compatible with Qpatch II technology. MaxCyte electroporated assay-ready cells can be generated within three days post transfection. Cryopreserved assay-ready cells can be assayed on the Qpatch II immediately after thawing with no significant decrease in performance. The compatibility of MaxCyte electroporated assay-ready cells with the QPatch II system enables researchers to efficiently generate and screen ion channel-expressing cells without stable cell line generation.
Summary
- MaxCyte electroporation efficiently transfects cell lines, primary human cells and stem cells.
- Transient transfection with MaxCyte electroporation enables simplified expression of complex, multi-subunit or toxic proteins.
- MaxCyte electroporation enables scalability from assay development to compound library screening without re-optimization.
- Transiently transfected, assay-ready cells are a suitable alternative to stable cell lines for ion channel research, assay development and drug discovery, decreasing reliance on costly stable cell lines.
Related resources
References
- Smith E, Dukovski D, Shumate J, Scampavia L, Miller JP, Spicer TP. Identification of Compounds That Promote Readthrough of Premature Termination Codons in the CFTR. SLAS Discov. 2021 Feb;26(2):205-215. doi: 10.1177/2472555220962001. Epub 2020 Oct 5. PMID: 33016182; PMCID: PMC7838340.
- Vanoye CG, Desai RR, Ji Z, Adusumilli S, Jairam N, Ghabra N, Joshi N, Fitch E, Helbig KL, McKnight D, Lindy AS, Zou F, Helbig I, Cooper EC, George AL Jr. High-throughput evaluation of epilepsy-associated KCNQ2 variants reveals functional and pharmacological heterogeneity. JCI Insight. 2022 Mar 8;7(5):e156314. doi: 10.1172/jci.insight.156314.
